Molecular Surveillance Studies Research Articles

First report and genome sequencing of SARS-CoV-2 in a cat (Felis catus) in Colombia.

BACKGROUND Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a virus of zoonotic origin that can bind to ACE2 receptors on the cells of many wild and domestic mammals. Studies have shown that the virus can circulate among animals mutate, lead to animal-to-human zoonotic jump, and further onward spread between humans. Infection in pets is unusual, and there are few human-to-pet transmission reports worldwide.OBJECTIVE To describe the SARS-CoV-2 infection in a domestic animal in Córdoba, Colombian Caribbean region.METHODS A cross-sectional molecular surveillance study was carried out, oral and rectal swabs were taken from cats and dogs living with people diagnosed with coronavirus disease 2019 (COVID-19).RESULTS SARS-CoV-2 was found in a cat living with a person with COVID-19. Genome sequencing showed that the B.1.111 lineage caused the infection in the cat. The owner’s sample could not be sequenced. The lineage is predominant in Colombia, and this variant is characterised by the presence of the D614D and Q57H mutation.CONCLUSION The present work is the first report of an infected cat with SARS-CoV-2 with whole-genome sequencing in Colombia. It highlights the importance of detecting SARS-CoV-2 mutations that could promote the transmissibility of this new coronavirus. There is still a significant information gap on human-to-cat-to-human infection; we encourage self-isolation measures between COVID-19 patients and companion animals. The findings of this study give a preliminary view of the current panorama of SARS-CoV-2 infection in animals in Colombia.

COVID-19 infections in pets such as cats and dogs

Coronaviruses are a very large group of virus families that can cause diseases in both different animal species and humans. Infections with different coronaviruses are seen in cats and dogs. The epidemic due to COVID-19 infection and its etiological agent SARS-CoV-2, which is a coronavirus, continues to spread very rapidly all over the world. In this article, we aimed to compile published studies on COVID-19 infections in cats and dogs, which we are in close contact with. When the studies were examined, it was understood that cats were more sensitive to SARS-CoV-2 than dogs after contact with COVID-19 positive individuals in quarantine. Cats may show symptoms in case of infection, compared to dogs. The RBD region where SARS-CoV-2 binds to ACE2 receptors is reported more similar to humans in cats than dogs. ACE2 protein seemed very similar to humans in both cats and dogs compared to other mammals. ACE2 receptor is highly expressed in different tissues of them, according to the study data examined. In a conclusion, SARS-CoV-2 can be detected because ACE2 is frequently expressed in different tissues in domestic animals such as cats and dogs, and the binding site is similar to human ACE2 receptors. In order to prevent zoonotic infections such as COVID-19 infections during the epidemic period when different disciplines need to work together with a single health approach, we believe that molecular surveillance studies should be carried out on our close friends such as cats and dogs. The data should be presented in public database and followed.

HIV-1 subtypes and profiles of resistance to protease inhibitors in the Marajo Archipelago (Brazilian Amazon region)

The Marajo Archipelago, located in the rural area of ​​the state of Para (Amazon region of Brazil), is the region with the lowest index of human development in the country, presenting poor health services, which makes adequate epidemiological surveillance of HIV-1 infection difficult. After a serological screening enrolling 1,877 samples, three samples were diagnosed with HIV-1 and the pro gene was sequenced to evaluate the presence of resistance mutations to protease inhibitors. Molecular analyses revealed, for the first time in the region of Marajo Island, the occurrence of HIV-1 subtypes B and D, as well as the presence of transmitted high and intermediate protease inhibitors resistance mutations. The results emphasize the importance of ongoing molecular epidemiological surveillance studies in the Brazilian Amazon region because antiretroviral (ARV) resistance mutations may limit treatment options, and the presence of certain subtypes seems to influence the progression to AIDS, particularly in areas where the entry and spread of the virus can be facilitated by socio-demographic problems that expose the population to sexually transmitted infections.

IN SILICO ANALYSIS OF RESISTANCE GENE IDENTIFIERS ON PLASMIDS OF BLANDM-5 PRODUCING CARBAPENEM-RESISTANT ESCHERICHIA COLI STRAINS ISOLATED IN HUMANS AND ANIMALS

Due to their ability to resist endpoint antimicrobials such as carbapenem, it is very important to detect and monitor multi-drug resistant Gram negative strains with plasmids containing genes such as blaNDM-5 by new molecular methods. This study aimed to perform in silico analysis of resistance gene identifiers on human and animal-derived blaNDM-5 plasmids found in open databases, which were analyzed by new whole genome sequencing techniques and to compare these resistance genes. The plasmid genomic sequences of 4 human and 2 animal E. coli strains containing blaNDM-5 genes included in our study were analyzed in Silico using the Resistance Gene Identifier (RGI) option of the comprehensive antibiotic resistance gene database using default values. Human and animal strains included in our study were found to have different antimicrobial resistance genes in addition to blaNDM-5. All plasmids were found to have at least 8 perfect antimicrobial resistance gene sequences matches. When the resistance gene identifiers in all plasmids were examined, 35 resistance gene identifiers were found. Besides blaNDM-5, mphA, qacEdelta1 and sul1 were found in all plasmids. As a conclusion, it was determined by our study results that, regardless of the source, there may be different antimicrobial resistance gene identifiers besides the blaNDM-5 resistance gene in plasmids. We are of the opinion that routine molecular surveillance studies should be carried out considering the one health approach of Gram-negative pathogens such as E. coli, which can contain plasmids that cause multi-drug resistance and can be isolated from all sources.

Genetic Characterization of a New HIV-1 Sub-Subtype A in Cabo Verde, Denominated A8.

Previous molecular characterization of Human immunodeficiency virus (HIV-1) samples from Cabo Verde pointed out a vast HIV-1 pol diversity, with several subtypes and recombinant forms, being 5.2% classified as AU-pol. Thus, the aim of the present study was to improve the characterization of these AU sequences. The genomic DNA of seven HIV-1 AU pol-infected individuals were submitted to four overlapping nested-PCR fragments aiming to compose the full-length HIV-1 genome. The final classification was based on phylogenetic trees that were generated using the maximum likelihood and bootscan analysis. The genetic distances were calculated using Mega 7.0 software. Complete genome amplification was possible for two samples, and partial genomes were obtained for the other five. These two samples grouped together with a high support value, in a separate branch from the other sub-subtypes A and CRF26_A5U. No recombination was verified at bootscan, leading to the classification of a new sub-subtype A. The intragroup genetic distance from the new sub-subtype A at a complete genome was 5.2%, and the intergroup genetic varied from 8.1% to 19.0% in the analyzed fragments. Our study describes a new HIV-1 sub-subtype A and highlights the importance of continued molecular surveillance studies, mainly in countries with high HIV molecular diversity.

Characterization of an Infectious Bronchitis Virus Isolated from Commercial Layers Suffering from False Layer Syndrome.

Infectious bronchitis virus (IBV) is a gammacoronavirus that primarily induces an upper respiratory disease in chickens, also affecting the urogenital tract and occasionally leading to a condition called false layer syndrome (FLS), where sexually mature hens ovulate normally but are unable to lay eggs. Here, we describe an outbreak of FLS in Arizona from which an IBV variant that is nearly 90% homologous to DMV/1639 using the Spike subunit 1 gene, named AZ/FLS/17, was isolated and used in challenge experiments. Three-day-old specific-pathogen-free chicks were challenged with AZ/FLS/17 or M41 in high and low doses, and the disease outcomes were compared. Overall, no differences in microscopic lesions or viral loads in the reproductive tract were detected between AZ/FLS/17- and M41-infected birds. To minimize the losses linked to FLS in the problematic flocks, an updated live-attenuated IBV vaccine protocol including the use of the Ma5 strain at the hatchery was implemented, resulting in a drastic reduction of false layers in the subsequent flocks. To monitor the circulation of wild-type and vaccine strains in this population, a molecular surveillance study was performed. Samples were collected at 1, 7, 14, and 21 days of age, and from laying hens at 30 and 36 wk. In older birds, the IBV strains detected were more diverse than at 1 and 7 days of age. Nevertheless, live vaccine combinations are still widely used to decrease the losses caused by FLS in commercial egg laying flocks worldwide.

Burden, Antibiotic Resistance, and Clonality of Shigella spp. Implicated in Community-Acquired Acute Diarrhoea in Lilongwe, Malawi.

Although numerous studies have investigated diarrhoea aetiology in many sub-Saharan African countries, recent data on Shigella species’ involvement in community-acquired acute diarrhoea (CA-AD) in Malawi are scarce. This study investigated the incidence, antibiotic susceptibility profile, genotypic characteristics, and clonal relationships of Shigella flexneri among 243 patients presenting with acute diarrhoea at a District Hospital in Lilongwe, Malawi. Shigella spp. were isolated and identified using standard microbiological and serological methods and confirmed by identifying the ipaH gene using real-time polymerase chain reaction. The isolates’ antibiotic susceptibility to 20 antibiotics was determined using the VITEK 2 system according to EUCAST guidelines. Genes conferring resistance to sulfamethoxazole (sul1, sul2 and sul3), trimethoprim (dfrA1, dfrA12 and dfrA17) and ampicillin (oxa-1 and oxa-2), and virulence genes (ipaBCD, sat, ial, virA, sen, set1A and set1B) were detected by real-time PCR. Clonal relatedness was assessed using ERIC-PCR. Thirty-four Shigella flexneri isolates were isolated (an overall incidence of 14.0%). All the isolates were fully resistant to sulfamethoxazole/trimethoprim (100%) and ampicillin (100%) but susceptible to the other antibiotics tested. The sul1 (79%), sul2 (79%), sul3 (47%), dfrA12 (71%) and dfrA17 (56%) sulfonamide and trimethoprim resistance genes were identified; Oxa-1, oxa-2 and dfrA1 were not detected. The virulence genes ipaBCD (85%), sat (85%), ial (82%), virA (76%), sen (71%), stx (71%), set1A (26%) and set1B (18%) were detected. ERIC-PCR profiling revealed that the Shigella isolates were genetically distinct and clonally unrelated, indicating the potential involvement of genetically distinct S. flexneri in CA-AD in Malawi. The high percentage resistance to ampicillin and sulfamethoxazole/trimethoprim and the presence of several virulence determinants in these isolates emphasises a need for continuous molecular surveillance studies to inform preventive measures and management of Shigella-associated diarrhoeal infections in Malawi.

Global evolutionary epidemiology and resistome dynamics of Citrobacter species, Enterobacter hormaechei, Klebsiella variicola, and Proteeae clones.

Citrobacter spp., Enterobacter hormaechei subsp., Klebsiella variicola and Proteae tribe members are rarely isolated Enterobacterales increasingly implicated in nosocomial infections. Herein, we show that these species contain multiple genes encoding resistance to important antibiotics and are widely and globally distributed, being isolated from human, animal, plant, and environmental sources in 67 countries. Certain clones and clades of these species were internationally disseminated, serving as reservoirs and mediums for the global dissemination of antibiotic resistance genes. As they can easily transmit these genes to more pathogenic species, additional molecular surveillance studies should be undertaken to identify and contain these antibiotic-resistant species.

Identification of a New HIV-1 BC Intersubtype Circulating Recombinant Form (CRF108_BC) in Spain.

The extraordinary genetic variability of human immunodeficiency virus type 1 (HIV-1) group M has led to the identification of 10 subtypes, 102 circulating recombinant forms (CRFs) and numerous unique recombinant forms. Among CRFs, 11 derived from subtypes B and C have been identified in China, Brazil, and Italy. Here we identify a new HIV-1 CRF_BC in Northern Spain. Originally, a phylogenetic cluster of 15 viruses of subtype C in protease-reverse transcriptase was identified in an HIV-1 molecular surveillance study in Spain, most of them from individuals from the Basque Country and heterosexually transmitted. Analyses of near full-length genome sequences from six viruses from three cities revealed that they were BC recombinant with coincident mosaic structures different from known CRFs. This allowed the definition of a new HIV-1 CRF designated CRF108_BC, whose genome is predominantly of subtype C, with four short subtype B fragments. Phylogenetic analyses with database sequences supported a Brazilian ancestry of the parental subtype C strain. Coalescent Bayesian analyses estimated the most recent common ancestor of CRF108_BC in the city of Vitoria, Basque Country, around 2000. CRF108_BC is the first CRF_BC identified in Spain and the second in Europe, after CRF60_BC, both phylogenetically related to Brazilian subtype C strains.

Multilocus Sequence Typing Reveals Clonality of Fluconazole-Nonsusceptible Candida tropicalis: A Study From Wuhan to the Global.

Candida tropicalis is a globally distributed human pathogenic yeast, and its increasing resistance to azoles makes clinical treatment difficult. In this study, we investigated the clinical features, azole resistance and genetic relatedness of 87 C. tropicalis isolates from central China and combined with the global database to explore the relationship between genetic information and fluconazole susceptibility. Of the 55 diploid sequence types (DSTs) identified by multilocus sequence typing (MLST), 27 DSTs were new to the C. tropicalis MLST database. Fluconazole-nonsusceptible (FNS) isolates were genetically closely related. goeBURST analysis showed that DST225, DST376, DST506, and DST546 formed a distinct and unique FNS clonal complex (CC) in Wuhan. The local FNS CC belongs to the large FNS CC (CC2) in China, in which the putative founder DST225 has been reported from the environment. The three most prevalent types (DST506, DST525, and DST546) in Wuhan had high minimum inhibitory concentrations (MICs) for antifungal azoles, and the six possible nosocomial transmissions we captured were all FNS strains, most of which were from CC2. Unique FNS CCs have been found in Singapore (CC8) and India (CC17) and are close to China’s CC2 in the minimum spanning tree. There were no FNS CCs outside Asia. This study is the first to reveal a significant correlation between genetic information and fluconazole susceptibility worldwide and to trace geographical locations, which is of great value for molecular epidemiological surveillance and azole-resistance study of C. tropicalis globally.

Identification of Novel Astroviruses in the Gastrointestinal Tract of Domestic Cats

Astroviruses, isolated from numerous avian and mammalian species including humans, are commonly associated with enteritis and encephalitis. Two astroviruses have previously been identified in cats, and while definitive evidence is lacking, an association with enteritis is suggested. Using metagenomic next-generation sequencing of viral nucleic acids from faecal samples, we identified two novel feline astroviruses termed Feline astrovirus 3 and 4. These viruses were isolated from healthy shelter-housed kittens (Feline astrovirus 3; 6448 bp) and from a kitten with diarrhoea that was co-infected with Feline parvovirus (Feline astrovirus 4, 6549 bp). Both novel astroviruses shared a genome arrangement of three open reading frames (ORFs) comparable to that of other astroviruses. Phylogenetic analysis of the concatenated ORFs, ORF1a, ORF1b and capsid protein revealed that both viruses were phylogenetically distinct from other feline astroviruses, although their precise evolutionary history could not be accurately determined due to a lack of resolution at key nodes. Large-scale molecular surveillance studies of healthy and diseased cats are needed to determine the pathogenicity of feline astroviruses as single virus infections or in co-infections with other enteric viruses.

Diversity of L1/L2 genes and molecular epidemiology of high-level carbapenem resistance Stenotrophomonas maltophilia isolates from animal production environment in China.

Stenotrophomonas maltophilia is emerging as a significant cause of human and animal disease worldwide. A total of 3400 samples were collected from animal farms and adjacent environments in China. The blaL1 and blaL2 genes were identified using whole genome sequence analyses and examined by phylogenetics. Isolates were also tested for susceptibility to 18 antibiotics. We isolated 118 strains of S. maltophilia from 3400 samples. The positive rates of blaL1 and blaL2 genes were 75% (89/118) and 22% (26/118) and we identified 11 L1 and 6 L2 amino acid sequence variants. S. maltophilia has at least two inducible β-lactamases (L1 and L2) that can hydrolyze almost all classes of β-lactams and these genes are suspected to confer carbapenem resistance. This represents a significant public health threat especially for hospitalized patients. We conducted a molecular surveillance study on the prevalence and characteristics of the blaL1 and blaL2 genes of S. maltophilia.

Reconsidering Mycobacterium bovis as a proxy for zoonotic tuberculosis: a molecular epidemiological surveillance study

SummaryBackgroundZoonotic tuberculosis is defined as human infection with Mycobacterium bovis. Although globally, India has the largest number of human tuberculosis cases and the largest cattle population, in which bovine tuberculosis is endemic, the burden of zoonotic tuberculosis is unknown. The aim of this study was to obtain estimates of the human prevalence of animal-associated members of the Mycobacterium tuberculosis complex (MTBC) at a large referral hospital in India.MethodsWe did a molecular epidemiological surveillance study of 940 positive mycobacteria growth indicator tube (MGIT) cultures, collected from patients visiting the outpatient department at Christian Medical College (Vellore, India) with suspected tuberculosis between Oct 1, 2018, and March 31, 2019. A PCR-based approach was applied to subspeciate cultures. Isolates identified as MTBC other than M tuberculosis or as inconclusive on PCR were subject to whole-genome sequencing (WGS), and phylogenetically compared with publicly available MTBC sequences from south Asia. Sequences from WGS were deposited in the National Center for Biotechnology Information Sequence Read Archive, accession number SRP226525 (BioProject database number PRJNA575883).FindingsThe 940 MGIT cultures were from 548 pulmonary and 392 extrapulmonary samples. A conclusive identification was obtained for all 940 isolates; wild-type M bovis was not identified. The isolates consisted of M tuberculosis (913 [97·1%] isolates), Mycobacterium orygis (seven [0·7%]), M bovis BCG (five [0·5%]), and non-tuberculous mycobacteria (15 [1·6%]). Subspecies were assigned for 25 isolates by WGS, which were analysed against 715 MTBC sequences from south Asia. Among the 715 genomes, no M bovis was identified. Four isolates of cattle origin were dispersed among human sequences within M tuberculosis lineage 1, and the seven M orygis isolates from human MGIT cultures were dispersed among sequences from cattle.InterpretationM bovis prevalence in humans is an inadequate proxy of zoonotic tuberculosis. The recovery of M orygis from humans highlights the need to use a broadened definition, including MTBC subspecies such as M orygis, to investigate zoonotic tuberculosis. The identification of M tuberculosis in cattle also reinforces the need for One Health investigations in countries with endemic bovine tuberculosis.FundingBill & Melinda Gates Foundation, Canadian Institutes for Health Research.

Molecular surveillance of methicillin-susceptible Staphylococcus aureus (MSSA) isolated over a one-year period from a Malaysian Teaching Hospital.

We report the results of a molecular surveillance study carried out on methicillin-susceptible Staphylococcus aureus (MSSA) isolated in a one-year duration from Hospital Canselor Tuanku Muhriz (HCTM), a tertiary hospital located in Kuala Lumpur, Malaysia. The first strain isolated from each MSSA infection in HCTM during the year 2009 was included into the study. Antimicrobial susceptibility testing and agr group typing were carried out for all strains; virulence gene (cna, seh, TSST-1 and PVL) typing results of the strains were obtained from a previous study. Pulsed-field gel electrophoresis (PFGE) was done on selected strains from the orthopedic ward. Relationship(s) between different typing methods used in the study was investigated, where a p value of <0.05 indicated significant association between typing methods. A total of 880 MSSA strains were included into the study. The strains were generally susceptible to most antibiotics, with most of them carrying cna and agr-I (51.6%, n=454; 39.8%, n=350, respectively). A total of 17 PFGE pulsotypes were identified using an 80% similarity cut-off value, where the main pulsotype (pulsotype E) consisted of 24 isolates (23.5%). agr-III strains were found to be usually positive for both cna and seh (p<0.05). In addition, some PFGE pulsotypes were also characteristic of certain virulence genes or agr groups. We did not identify a dominant MSSA clone circulating in HCTM in 2009. Nevertheless, results from this molecular surveillance will provide good baseline data for the hospital's second S. aureus surveillance planned for the year 2020.

Animal Disease Surveillance in the 21st Century: Applications and Robustness of Phylodynamic Methods in Recent U.S. Human-Like H3 Swine Influenza Outbreaks.

Emerging and endemic animal viral diseases continue to impose substantial impacts on animal and human health. Most current and past molecular surveillance studies of animal diseases investigated spatio-temporal and evolutionary dynamics of the viruses in a disjointed analytical framework, ignoring many uncertainties and made joint conclusions from both analytical approaches. Phylodynamic methods offer a uniquely integrated platform capable of inferring complex epidemiological and evolutionary processes from the phylogeny of viruses in populations using a single Bayesian statistical framework. In this study, we reviewed and outlined basic concepts and aspects of phylodynamic methods and attempted to summarize essential components of the methodology in one analytical pipeline to facilitate the proper use of the methods by animal health researchers. Also, we challenged the robustness of the posterior evolutionary parameters, inferred by the commonly used phylodynamic models, using hemagglutinin (HA) and polymerase basic 2 (PB2) segments of the currently circulating human-like H3 swine influenza (SI) viruses isolated in the United States and multiple priors. Subsequently, we compared similarities and differences between the posterior parameters inferred from sequence data using multiple phylodynamic models. Our suggested phylodynamic approach attempts to reduce the impact of its inherent limitations to offer less biased and biologically plausible inferences about the pathogen evolutionary characteristics to properly guide intervention activities. We also pinpointed requirements and challenges for integrating phylodynamic methods in routine animal disease surveillance activities.

Application of Ribosomal Internal Transcribed Spacer 1, Internal Transcribed Spacer 2, and Large-Subunit D1-D2 Regions as the Genetic Markers to Identify Fungi Isolated from Different Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

In September 2012, a multistate fungal meningitis outbreak started across 20 states in the United States. It affected 753 individuals and caused 64 deaths who received contaminated spinal injections. In a previous study, we analyzed 26 environmental samples collected from the manufacturing premises of a compounding company to determine the possible cause of an outbreak and identified 14 distinct fungal species. In this follow-up study, we have analyzed 198 environmental samples collected from three additional compounding company premises located in the United States for the presence of pathogenic fungi. Environmental swab samples were initially examined by standard microbiological methods. Subsequently, DNA sequencing was performed on all of the 25 recovered fungal isolates at the D1-D2 domain of the large subunit (LSU) ribosomal RNA (rRNA) and the internal transcribed spacer (ITS) regions. Sequence analysis of the ITS1, ITS2, and LSU rRNA regions confirmed the presence of the following fungal species in the environmental samples analyzed: (i) Pestalotiopsis cocculi from the region Ia; (ii) Epicoccum nigrum and Trichaptum biforme from the region Ib; (iii) Nigrospora sphaerica and Fusarium sp. from the region II; and (iv) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Species identification of 25 recovered fungal isolates matched, in most cases, at 3 sequenced loci (ITS1, ITS2, and LSU). DNA sequencing of ITS1, ITS2, and LSU D1-D2 regions can be used to perform fungal typing and in implementing effective environmental monitoring programs of public health importance.

Whole-genome sequence-based comparison and profiling of virulence-associated genes of Bacillus cereus group isolates from diverse sources in Japan

BackgroundThe complete genome sequences of 44 Bacillus cereus group isolates collected from diverse sources in Japan were analyzed to determine their genetic backgrounds and diversity levels in Japan. Multilocus sequence typing (MLST) and core-genome single-nucleotide polymorphism (SNP) typing data from whole-genome sequences were analyzed to determine genetic diversity levels. Virulence-associated gene profiles were also used to evaluate the genetic backgrounds and relationships among the isolates.ResultsThe 44 B. cereus group isolates, including soil- and animal-derived isolates and isolates recovered from hospitalized patients and food poisoning cases, were genotyped by MLST and core-genome SNP typing. Genetic variation among the isolates was identified by the MLST and core-genome SNP phylogeny comparison against reference strains from countries outside of Japan. Exploratory principal component analysis and nonmetric multidimensional scaling (NMDS) analyses were used to assess the genetic similarities among the isolates using gene presence and absence information and isolate origins as the metadata. A significant correlation was seen between the principal components and the presence of genes encoding hemolysin BL and emetic genetic determinants in B. cereus, and the capsule proteins in B. anthracis. NMDS showed that the cluster of soil isolates overlapped with the cluster comprising animal-derived and clinical isolates.ConclusionsMolecular and epidemiological analyses of B. cereus group isolates in Japan suggest that the soil- and animal-derived bacteria from our study are not a significant risk to human health. However, because several of the clinical isolates share close genetic relationships with the environmental isolates, both molecular and epidemiological surveillance studies could be used effectively to estimate virulence in these important pathogens.

High colonization rate of a novel carbapenem-resistant Klebsiella lineage among migratory birds at Qinghai Lake, China.

The emergence of carbapenemase-positive Enterobacteriaceae poses a serious threat to public health worldwide. Here we conducted a molecular surveillance study on carbapenem-resistant Enterobacteriaceae (CRE) colonization among migratory birds at Qinghai Lake in China. A total of 420 samples from migratory birds and their surrounding environment were collected at three sites along the Qinghai Lake bird island. Carbapenem-non-susceptible isolates were identified by 16S rDNA sequencing and MALDI-TOF MS. Carbapenemase producers were determined by Carba NP testing. Antimicrobial susceptibility testing, transfer ability and PFGE were also performed, and 46 isolates from different pulsotypes were analysed by WGS. Three hundred and fifty isolates were carbapenemase producers based on Carba NP testing, while 233 Klebsiella spp. and 2 Escherichia coli isolates were NDM-5-carriers. PFGE was performed and showed that the isolates were grouped into five pulsotypes; among these, type A was predominant (86.7%, n = 202) and belonged to a novel Klebsiella lineage, ST1697. WGS analysis indicated that ST1697 strains may be a hybrid of the recombination of Klebsiella quasipneumoniae subsp. similipneumoniae and Klebsiella pneumoniae genomes. This high frequency of carbapenemase producers in migratory birds is unexpected. These results provide new insight into the spread of antibiotic resistance, and highlight that continued vigilance for MDR carbapenemase-producing Enterobacteriaceae in migratory birds is urgently needed.

IncC blaKPC-2-positive plasmid characterised from ST648 Escherichia coli.

This study describes the characterisation of type 2 IncC plasmids pC-Ec20-KPC and pC-Ec2-KPC, carrying theblaKPC-2 gene, from two multiresistant Escherichia coli recovered in University Hospital of Larissa (Greece) in 2018. E. coli strains Ec-2Lar and Ec-20Lar were recovered from rectal swabs of two patients during monthly surveillance cultures. Transfer experiments by conjugation were carried out using rifampicin-resistant E. coli A15 laboratory strain as recipient. blaKPC-carrying plasmids were characterised by S1 profiling. Isolates were typed by MLST. Whole-genome sequencing was performed using the Sequel platform. Both E. coli isolates, belonging to ST648, transferred blaKPC-2 to E. coli A15 by conjugation. Plasmid analysis revealed that the transconjugants harboured blaKPC-positive plasmids of different sizes. Analysis of plasmid sequences showed that in both isolates the blaKPC-2 gene was carried on a type 2 IncC plasmid (pC-Ec20-KPC and pC-Ec2-KPC, respectively). Both plasmids carried the ARI-B resistance island consisting of several resistance genes, intact and truncated copies of several mobile elements, and a 25 571-bp segment harbouring coding sequences for an iron transporter. The blaKPC-2 gene was part of transposon Tn4401a, which was bounded by 5-bp direct repeats (TCCTT) suggesting its transposition into the IncC plasmids. To our knowledge, this is the first report on complete nucleotide sequences of type 2 IncC plasmids. These findings, which hypothesise the acquisition of KPC-2-encoding transposon Tn4401a by an IncC replicon, indicate the ongoing need for molecular surveillance studies of multidrug-resistant pathogens. In addition, they underline the increasing clinical importance of the IncC plasmid family.

Genetic Characterization of Fungi Isolated from Environmental Samples: A Molecular Surveillance Study of Public Health Importance.

Background: A multistate fungal meningitis outbreak began in September 2012 that affected 751 individuals who received contaminated spinal injections across 20 states in the United States, which led to 64 deaths. In our previous study, we examined 26 environmental swab samples collected from various locations of the manufacturing premises of the compounding company to determine the possible cause of this outbreak and identified 14 novel species of fungi. Objective: In this follow-up study, a total of 198 environmental surveillance samples were collected and analyzed to detect pathogenic fungal species from other compounding company premises located in three regions of the United States. Methods: DNA sequencing was performed at the large subunit ribosomal RNA (LSU rRNA) and internal transcribed spacer (ITS) regions on the 25 positive fungal isolates. Results: Sequence analysis of the ITS1, the ITS2, and the LSU rRNA regions confirmed the presence of the following fungal species in the samples analyzed: (1) Pestalotiopsis cocculi from the region I; (2) Epicoccum nigrum and Trichaptum biforme from the region I; (3) Nigrospora sphaerica and Fusarium sp. from the region II; and (4) Curvularia sp., Fusarium sp., Penicillium sp., and Preussia sp. from the region III. Conclusions: Our results suggest that the LSU and ITS regions are good genetic markers to perform fungal typing. Highlights: DNA sequencing technology can be used in the implementation of effective environmental monitoring programs of public health importance.